Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or beta-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10-7 g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium, bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Phylogenetic systematics of Sternbergia (Amaryllidaceae) based on plastid and ITS sequence data

The phylogenetics of Sternbergia (Amaryllidaceae) were studied using DNA sequences of the plastid ndhF and matK genes and nuclear internal transcribed spacer (ITS) ribosomal region for 38, 37 and 32 ingroup and outgroup accessions, respectively. All members of Sternbergia were represented by at least one accession, except S. minoica and S. schubertii, with additional taxa from Narcissus and Pancratium serving as principal outgroups. Sternbergia was resolved and supported as sister to Narcissus and composed of two primary subclades: S. colchiciflora sister to S. vernalis, S. candida and S. clusiana, with this clade in turn sister to S. lutea and its allies in both Bayesian and bootstrap analyses. A clear relationship between the two vernal flowering members of the genus was recovered, supporting the hypothesis of a single origin of vernal flowering in Sternbergia. However, in the S. lutea complex, the DNA markers examined did not offer sufficient resolving power to separate taxa, providing some support for the idea that S. sicula and S. greuteriana are conspecific with S. lutea

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayestan phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes Within the Albimanus Section the monophyly of the Stroder Subgroup was strongly supported and within the Myzorhynchella Section Anopheles anrunesi and An lutzu formed a strongly supported monophyletic group The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An lanet-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An goeldii were recovered as sister species Finally, there was evidence of complexes in several species, including An antunesi, An deaneorum, and An. strodei (c) 2010 Elsevier B.V. All rights reserved

Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae) Species from Brazil

Anopheles (Nyssorhynchus) benarrochi s.l., Anopheles (Nyssorhynchus) oswaldoi s.l., and Anopheles (Nyssorhynchus) konderi s.l. collected in Acrelandia, state of Acre, Brazil, were identified based on morphological characters of the male genitalia, fourth-instar larvae, and pupae. Morphological variation was observed in the male genitalia of these species in comparison with specimens from other localities in Brazil. DNA sequence from the nuclear ribosomal second internal transcribed spacer of individuals identified as An. benarrochi s.l. by using male genitalia characteristics showed that the various morphological forms are conspecific but are distinct from An. benarrochi B from Colombia. Anopheles konderi s.l. and An. oswaldoi s.l. both misidentified as An. oswaldoi s.s. (Peryassu) throughout Brazil, may actually comprise at least two undescribed species. Diagnostic morphological characteristics of the male genitalia are provided to distinguish Anopheles benarrochi s.l., Anopheles oswaldoi s.l., and Anopheles konderi s.l. from morphologically similar species. Incrimination of An. oswaldoi s.s. in malaria transmission in Brazil needs further investigation because other undescribed species from Acre may have been confounded with this taxon.

Evidence that the Ceratobasidium-like white-thread blight and black rot fungal pathogens from persimmon and tea crops in the Brazilian Atlantic Forest agroecosystem are two distinct phylospecies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cryptic hammerhead shark lineage occurrence in the western South Atlantic revealed by DNA analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Streamlined DNA Tool for Global Identification of Heavily Exploited Coastal Shark Species (Genus Rhizoprionodon)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxacillin Resistance and Antimicrobial Susceptibility Profile of Staphylococcus saprophyticus and Other Staphylococci Isolated from Patients with Urinary Tract Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Análise filogenética de Phytophthora capsici Leonian do estado de São Paulo baseada na seqüência de nucleotídeos da região ITS-5.8S rDNA

Phylogenetic analysis through morphological and cultural traits is considered inconsistent and hard to be scientifically accepted once there is no way to establish the direction of evolution on morphological traits. Molecular markers are suitable for phylogenetic analysis. The sequencing of ITS1 and ITS2 (internal transcribed spacers) regions and of 5.8S gene from ribosomal DNA were used to estimate the genetic variation and distance of 10 Phytophthora capsici isolates. The amount of genetic variation amongst isolated P. capsici from distinct regions of São Paulo State was 0.1 to 1.6 and 0.1 to 1.1% at ITS1 and ITS2, respectively, and a phylogenetic distance of about 78.5% between P. capsici and Phytophthora spp. was observed.

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion. © The Mycological Society of Japan and Springer-Verlag 2005.

Intraspecific variation and emendation of Hannaella kunmingensis

In order to investigate the intraspecific variability in Hannaella kunmingensis, 11 isolates, including the type strain, were analyzed for their morphological and biochemical traits. The combined internal transcribed spacer region (ITS), D1/D2 domains of the large subunit rDNA (LSU), and cytochrome b gene were examined using phylogenetic and parsimony network analyses. Our investigations revealed differences in colony morphology as well as differences in 31 out of 64 phenotypic characteristics examined, including growth in lactose, vitamin free medium, xylitol, L-arabinitol, and nitrite. Growth in the presence of 0. 1 % cycloheximide was also highlighted in H. kunmingensis. All the 11 strains were conspecific in the LSU; however, variations of about 2. 5 % were found in the ITS while isolate CBS 8356 exhibited a 27. 3 % divergence from the other strains in the cytochrome b gene. Parsimony network analysis revealed the existence of three haplotypes among the H. kunmingensis strains studied but excluded CBS 8356 from the network connecting these haplotypes. This study contributes to the knowledge of the intraspecific diversity of H. kunmingensis. To accommodate such intraspecific variations, an emendation of the species diagnosis is proposed. © 2012 German Mycological Society and Springer.

Fungal communities in the garden chamber soils of leaf-cutting ants

Leaf-cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant-altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf-cutting ants in Brazil, were determined and compared with non-nest soils. Culture-dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non-nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non-nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non-nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf-cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non-nest soils. © 2013 WILEY-VCH Verlag GmbH & Co.